There are many checkpoints that inhibit cell cycle progression in response to defects in inheritance of the nucleus including checkpoints that monitor nuclear DNA duplication, quality control and segregation. We identified a checkpoint that responds to defects in organelle inheritance, and more specifically to loss of mitochondrial DNA (mtDNA). The checkpoint for damage to nuclear DNA does not respond to loss of specific genes encoded by nuclear DNA. Rather, it responds to broader issues, including double strand DNA breaks or stalled replication forks. We find that the mtDNA inheritance checkpoint also does not respond to loss of genes encoded by mtDNA. Rather, it responds to loss of DNA in the organelle. All well-characterized checkpoints have 1) sensors that detect defects in critical cell cycle events, 2) signal transduction machinery to arrest the cell cycle and promote repair, and 3) mechanisms to inactivate the checkpoint. We obtained evidence for a role of Mip1p, mtDNA polymerase , and contact sites between mitochondrial outer and inner membranes in sensing loss of mtDNA. We also find that Rad53p, the yeast homologue of the tumor suppressor and the DNA damage checkpoint kinase Chk2, regulates G1 to S progression in response to loss of mtDNA. Our studies reveal the existence of an organelle inheritance checkpoint and raise the possibility that there are checkpoints for the inheritance of other organelles. Since mutation of Chk2 and its upstream (ATM) and downstream (p53) regulators also results in changes in mtDNA content and cell cycle delays in mammalian and human cells, this checkpoint likely exists in other eukaryotes. We will study how information regarding mtDNA content is transmitted from the mitochondrial inner membrane (where mtDNA and Mip1p reside) across contact sites to the cytosol and ultimately to the nucleus (where Rad53p resides). Here, we will focus on the replisome, a protein complex that contains Mip1p, spans mitochondrial contact sites and binds directly to mtDNA, and on pathways to transmit signals from the replisome to Rad53p. Since DNA replication is template-dependent, cells that have lost mtDNA cannot replace it. Therefore, they must adapt to the checkpoint and resume cell cycling to survive. We will study the role of largely uncharacterized genes that we have implicated checkpoint adaptation and regulation of G1 to S progression. The proposed studies will be the first to determine the mechanism underlying the mtDNA inheritance checkpoint and adaptation to that checkpoint. They will also extend our understanding of proteins that interact with mtDNA, particularly at mitochondrial contact sites; mechanisms for regulation of G1 to S progression; and signal transduction in the DNA damage checkpoint. Finally, since the DNA damage checkpoint is the target for mutation in cancer, and mtDNA polymerase is the target for mutation in >125 diseases, the studies will extend our understanding of cancer biology and diseases associated with defects in mitochondrial function.